Hindawi

BioMed Research International

Volume 2018, Article ID 3086586, 6 pages
https://doi.org/10.1155/2018/3086586

Research Article
In Vitro Antifungal Susceptibility of Candida Species Isolated
from Iranian Patients with Denture Stomatitis

Saeid Mahdavi Omran ,1 Maryam Rezaei Dastjerdi,2 Maryam Zuashkiani,2

Vahid Moqarabzadeh,3 and Mojtaba Taghizadeh-Armaki 4
1
Infectious Diseases and Tropical Medicine Research Center, Department of Medical Parasitology and Mycology,
Babol University of Medical Sciences, Babol, Iran
2
Department of Prosthodontics, Faculty of Dentistry, Babol University of Medical Sciences, Babol, Iran
3
Department of Biostatistics, Faculty of Health, Student Research Committee, Mazandaran University of Medical Sciences, Sari, Iran
4
Department of Medical Mycology and Parasitology, School of Medicine, Babol University of Medical Sciences, Babol, Iran

Correspondence should be addressed to Mojtaba Taghizadeh-Armaki; mojtabataghizade@yahoo.com

Received 19 January 2018; Revised 21 March 2018; Accepted 15 April 2018; Published 16 May 2018

Academic Editor: Nobuo Kanazawa

Copyright © 2018 Saeid Mahdavi Omran et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.

Background. Candida-associated denture stomatitis (CADS) is a common fungal infection in people who wear dentures. The main
objective of this study was to make molecular identification of causative agents of CADS and in vitro antifungal susceptibility testing
(AFST) in the Iranian patients with denture stomatitis. Methods. A total of 134 Candida spp. were obtained from patients with
denture stomatitis. The Candida spp. were identified using a polymerase chain reaction-restriction fragment length polymorphism
(PCR-RFLP) involving the universal internal transcribed spacer (ITS1 and ITS4) primers, which were subjected to digestion with
MspI and BlnI restriction enzymes. The in vitro antifungal susceptibility of Candida spp. to fluconazole (FLC), terbinafine (TRB),
itraconazole (ITC), voriconazole (VRC), posaconazole (POS), ketoconazole (KET), amphotericin B (AMB), and caspofungin (CAS)
was evaluated using the Clinical and Laboratory Standards Institute M27-A3 and M27-S4 guidelines. Results. Overall, C. albicans
was the most commonly isolated species (𝑛 = 84; 62.6%), followed by C. glabrata (𝑛 = 23; 17.2%), C. tropicalis (𝑛 = 16; 12%), and C.
parapsilosis (𝑛 = 11; 8.2%). Posaconazole had the lowest geometric mean minimum inhibitory concentration (MIC) (0.03 𝜇g/ml),
followed by AMB (0.05 𝜇g/ml), ITC (0.08 𝜇g/ml), VRC (0.11 𝜇g/ml), CAS (0.12 𝜇g/ml), KET (0.15 𝜇g/ml), and FLC (0.26 𝜇g/ml).
Discussion. Our study showed that C. albicans was most prevalent in Iranian patients with CADS and was susceptible to both azoles
and amphotericin B. In addition, POS could be an appropriate alternative to the current antifungal agents used for the treatment
of CADS, as well as in the treatment of recurrent candidiasis.

1. Introduction In recent years, non-albicans Candida infections and

Candida-associated denture stomatitis (CADS) is a chronic
atrophic complication of the oral cavity that mainly affects
people who wear removable dentures [1]. Several evidence-
based studies have shown that Candida albicans is the main
etiological agent of denture stomatitis (DS), followed by C.
tropicalis, C. parapsilosis, and C. glabrata [2–4]. The early
diagnosis of pathogenic fungal agents and the determination
of their susceptibility to antifungal drugs are critical to
the treatment of the infection and to establish preventive
healthcare-associated strategies [5, 6].
antifungal resistant isolates have increased; thus, developing
a reliable diagnostic method is essential for the management
of candidiasis [7, 8].
A molecular-based method, such as polymerase chain
reaction-restriction fragment length polymorphism (PCR-
RFLP), is a promising technique that is used in the identi-
fication of pathogenic Candida spp. [9].
The management of CADS depends on a wide-ranging
treatment strategy [10], which includes detecting and erad-
icating possible significant risk factors, preventing a systemic
Candida infection, and reducing any associated discom-
fort [11, 12]. The use of oral formulations of antimicrobial
2 BioMed Research International
agents, such as amphotericin B (AMB), nystatin (NYS), and
miconazole (MIC), and systemic drugs, such as fluconazole
(FLC), voriconazole (VRC), posaconazole (POS), itracona-
zole (ITC), and ketoconazole (KET), has been shown to be
effective in the treatment of CADS [13–16]. Echinocandins,
such as caspofungin (CAS), are a class of antifungal drugs
that appear to be highly effective against all Candida spp.,
including those that are less sensitive or are resistant to FLC
and/or ITC [15]. However, previous studies have described
the recurrence and clinical relapse of CADS after treatment
[1, 17, 18]. Having sufficient information about the antifungal
susceptibility testing (AFST) of the Candida spp. involved
in CADS may help in the selection of alternative antifungal
treatments for recurrent oral candidiasis. In the current study,
we evaluated the in vitro AFST of a collection of molecularly
identified Candida spp. isolated from Iranian patients with
DS.
2. Materials and Methods
2.1. Sample Collection Process. After an examination of the
oral cavity, denture samples were obtained by scraping sterile
swabs across the inner surface of the denture. In a period
of 3 years (2013 to 2016), a total of 134 clinical isolates were
collected from 103 patients aged 53–86 years affected with
DS. All samples were streaked on the Sabouraud dextrose
agar (Merck, Darmstadt, Germany) and incubated at 35∘ C
for 7 days. All suspected colonies were detected by CHRO-
Magar Candida (CHROMagar, Paris, France) and PCR-RFLP
methods. Each isolate was preserved in the tryptic soy broth
(TSB) (Merck, Darmstadt, Germany) and then stored in the
culture collection of the Department of Medical Mycology,
Babol University of Medical Sciences, Iran.
2.2. Genomic DNA Extraction and PCR-RFLP. The total ge-
nomic DNA from the yeast was removed using the method
described by Yamada et al., which involved cell disruption
with glass beads followed by extraction with phenol–chloro-
form and precipitation with ethanol [19].
Oligonucleotide primer sequences including ITS1 (5󸀠 -
TCCGTAGGTGAACCTGCGG-3󸀠 ) and ITS4 (5󸀠 -TCCTCC-
GCTTATTGATATGC-3󸀠 ) were used in this study [20].
Amplification was performed on a thermal cycler (C1000;
Bio-Rad Laboratories, Inc.). The amplified products were
electrophoresed on 1.5% agarose gels containing 0.5 mg/ml
of ethidium bromide and then analyzed under UV light
using a gel-doc system (Bio-Rad, USA). The breakdown of
the amplified products involved the restriction enzymes BlnI
and/or MspI (Table 1). The digests of the PCR fragments were
electrophoresed on 1.5% agarose gels. In this study, C. albicans
ATCC 10231 and C. dubliniensis CBS 2747 were used as quality
control strains.
2.3. Antifungal Susceptibility Testing. The following anti-
fungal agents were evaluated: AMB (Bristol-Myers Squib,
Woerden, Netherlands), FLC, ITC, VRC, KET, and TRB
(Sigma-Aldrich, St. Louis, MO, USA), POS (Schering-Plough
Corp., Oss, Netherlands), and CAS (Pfizer, Capelle aan den
Ijssel, Netherlands). In vitro AFST was performed according
Table 1: The cutting size PCR-products of ITS region for different
Candida spp.subjected to digestion with MspI and BlnI restriction
enzymes.
Size (s) of restriction
Candida species Size of ITS1-ITS4, bp product (s), bp
MspI BlnI
C. albicans 535 297, 238 535
C. glabrata 871 557, 314
C. tropicalis 524 340, 184
C. parapsilosis 520 520
C. dubliniensis 535 297, 238 200–335
to the Clinical and Laboratory Standards Institute (CLSI)
M27-A3 and M27-S4 guidelines [21, 22]. Each antifungal
agent was prepared separately. The final concentration of
FLC ranged from 0.063 to 64 𝜇g/ml. The final concentrations
of AMB, ITC, VRC, POS, and KET ranged from 0.016 to
16 𝜇g/ml, while the final concentrations of CAS and TRB
were 0.008–8 𝜇g/ml and 0.12–128 𝜇g/ml, respectively. The
drugs were diluted in RPMI-1640 Medium (Sigma-Aldrich,
Darmstadt, Germany) and buffered to pH 7.0 with 0.165 M N-
morpholinepropanesulfonic acid (MOPS) (Sigma-Aldrich,
USA) and L-glutamine without bicarbonate to yield twofold
their final concentrations. The primary Candida spp. were
cultured on potato dextrose agar (PDA; Difco, Leeuwarden,
Netherlands) and incubated for 3 days at 35∘ C. Once mature
colonies were observed, a conidial inoculum was made using
a sterile saline solution. A spectrophotometer at 530 nm
was used to adjust the inoculum to a range of 2.5–5 ×
106 CFU/ml. The drug containing 96-well plastic microplates
was inoculated with this suspension and incubated at 35∘ C for
24–48 h. The minimum inhibitory concentrations (MICs) for
FLC, VRC, CAS, ITC, and POS were determined according to
the CLSI M27-A3 and M27-S4 guidelines [21, 22]. Isolates that
responded to ≤1 𝜇g/ml MIC for AMB were recognized as sus-
ceptible isolates according to the CLSI M27-S3 guideline [23].
The breakpoint was not determined for TRB; however, several
studies have reported resistance breakpoints ≥ 8 𝜇g/ml [24,
25]. The breakpoint values for KET were not defined by the
CLSI and, thus, the resistant breakpoint of ≥4 𝜇g/ml which
was determined by Mulu et al. (2013) was used [26]. Isolates
from C. krusei (ATCC 6258) and C. parapsilosis (ATCC
22019) were used as quality control strains.
2.4. Data and Statistical Analysis. The geometric mean (GM),
MIC50 , and MIC90 for the antifungal agents against Candida
spp. were calculated using EXCEL (Microsoft Office Excel
2003 SP3, Microsoft Corporation, Redmond, USA).
3. Results
C. albicans was the predominant species (𝑛 = 84; 62.6%),
followed by C. glabrata (𝑛 = 23; 17.2%), C. tropicalis (𝑛 = 16;
12%), and C. parapsilosis (𝑛 = 11; 8.2%). Table 2 summarizes
the GM of the MICs, the MIC ranges, MIC50 , and MIC90
for the antifungal drugs against all Candida isolates. The
BioMed Research International 3

Table 2: In vitro antifungal susceptibility of eight antifungal agents against 134 Candida spp. isolated from Candida-associated denture
stomatitis.
MIC 𝜇g/mL
Candida species/number of strains/antifungal drugs
MIC range MIC50 MIC90 GM
All Candida species (134)
FLC 0.016–16 0.125 8 0.26
ITC 0.016–16 0.064 0.5 0.08
VRC 0.016–4 0.125 0.5 0.11
AMB 0.016–2 0.064 0.25 0.05
CAS 0.008–2 0.125 0.5 0.12
TRB 2–≥128 128 >128 65.00
POS 0.032–0.5 0.016 0.125 0.03
KET 0.016–4 0.125 1 0.15
C. albicans (84)
FLC 0.016–16 0.064 2 0.09
ITC 0.016–0.5 0.032 0.5 0.04
VRC 0.032–0.25 0.064 0.25 0.08
AMB 0.008–0.25 0.032 0.25 0.03
CAS 0.008–1 0.064 0.5 0.08
TRB 2–≥128 128 128 96.68
POS 0.032–0.5 0.016 0.032 0.01
KET 0.016–2 0.125 1 0.09
C. glabrata (23)
FLC 0.25–≥16 4 64 5.24
ITC 0.125–0.5 0.25 0.5 0.26
VRC 0.125–2 0.25 0.5 0.24
AMB 0.032–0.5 0.032 0.5 0.07
CAS 0.008–2 0.5 1 0.51
TRB 8–>128 16 64 19.77
POS 0.125–0.5 0.125 0.5 0.19
KET 0.064–1 0.125 0.5 0.14
C. tropicalis (16)
FLC 0.125–≥16 0.125 4 0.42
ITC 0.016–16 0.016 4 0.27
VRC 0.016–4 0.032 2 0.08
AMB 0.016–2 0.125 1 0.17
CAS 0.008–1 0.032 0.125 0.05
TRB 4–≥128 64 128 53.81
POS 0.016–0.25 0.032 0.25 0.05
KET 0.032–4 0.25 2 0.23
C. parapsilosis (11)
FLC 0.25–4 0.25 2 0.46
ITC 0.125–8 0.25 4 0.46
VRC 0.25–2 0.25 1 0.41
AMB 0.016–2 0.25 1 0.19
CAS 0.008–2 0.25 1 0.41
TRB 4–≥128 128 128 49.74
POS 0.032–0.125 0.032 0.125 0.05
KET 0.125–2 0.125 1 0.25
FLC, fluconazole; ITC, itraconazole; VRC, voriconazole; AMB, amphotericin B; CAS, caspofungin; TRB, terbinafine; POS, posaconazole; KET, ketoconazole;
GM, geometric mean; MIC, minimum inhibition concentration; MIC50 and MIC90, concentration at which 50% and 90% of the strains were inhibited,
respectively.
4 BioMed Research International
GM of MICs for drugs across all strains was, in increas-
ing order, 0.03 𝜇g/ml (POS), 0.05 𝜇g/ml (AMB), 0.08 𝜇g/ml
(ITC), 0.11 𝜇g/ml (VRC), 0.12 𝜇g/ml (CAS), 0.15 𝜇g/ml (KET),
0.26 𝜇g/ml (FLC), and 65.00 𝜇g/mL (TRB). All C. albicans
isolates (100%) were found to be susceptible to AMB, VRC,
POS, KET, and ITC; however, 13 isolates (15.5%) were
resistant to FLC. All C. parapsilosis isolates (100%) were
susceptible to FLC, while only 4 isolates (17.4%) of C. glabrata
and 2 isolates (12.5%) of C. tropicalis were resistant to FLC.
The resistance rates for VRC of C. tropicalis, C. parapsilosis,
and C. glabrata were 18.7% (3/16), 8.6% (2/23), and 9.1%
(1/11), respectively. The ITC MICs for 6 isolates (37.5%) of
C. tropicalis and 4 isolates (36.4%) of C. parapsilosis were
≥1 𝜇g/ml. The resistance rates for AMB in C. tropicalis and C.
parapsilosis were 12.5% (2/16) and 45.5% (5/11), respectively.
Out of 134 isolates, 1 isolate of C. tropicalis (≥4 𝜇g/ml) was
resistant to KET. The resistance rates for CAS in C. glabrata,
C. tropicalis, and C. albicans were 56.5% (13/23), 9.1% (1/11),
and 2.3% (2/84), respectively. Overall, all Candida spp. had
the highest in vitro antifungal susceptibility to ITC, POS, and
CAS. However, Candida spp. showed a lack of susceptibility
to TRB.
4. Discussion
Dentures in the oral cavity are considered to be a reservoir
of Candida spp. and, thus, are a predisposing factor for DS
in patients, as well as a potential origin of reinfection [27].
CADS is an infection initiated by the oral colonization of
Candida spp.; the most frequently identified species is C. albi-
cans, although C. glabrata, C. guilliermondii, C. parapsilosis,
C. krusei, and C. tropicalis are less commonly seen [28, 29].
In agreement with other studies, our research found that C.
albicans, C. parapsilosis, C. tropicalis, and C. glabrata caused
CADS [30–32]. The recommended drug of choice to treat
CADS in patients without an underlying disease commonly
includes a NYS suspension or a clotrimazole tablet. However,
a topical application of an azole, such as FLC or ITC, can also
be used to prevent persistent or chronic fungal infections in
the patients [33, 34].
Several studies reported the emergence of antifungal
resistance to azoles, which has been associated with multiple
episodes of recurrence [16, 35–37]. In the current study, 15.5%
of C. albicans (13/84) was observed to be resistant to FLC.
In contrast with our data, Abaci and Haliki-Uztan (2011)
reported that 59.4% of C. albicans were resistant to FLC [24].
AMB, also used in the management of CADS, proved
effective against Candida spp. [1]. Besides, the findings
obtained in the present study were in agreement with the
results by Wingeter et al. (2007) [38] regarding the suscep-
tibility of oral Candida strains to AMB.
AMB-resistant non-albicans isolates were reported from
several previous studies [24, 39]. We also found that 12.5%
(2/16) of C. tropicalis and 45.5% (5/11) of C. parapsilosis
isolates showed resistance patterns to AMB. The good in vitro
activities of POS and VRC have been previously reported
against Candida spp. obtained from oral candidiasis patients
[40–43].
As shown in Table 2, POS was the most effective drug
in vitro with GM MICs of 0.01 𝜇g/ml, 0.19 𝜇g/ml, 0.05 𝜇g/ml,
and 0.05 𝜇g/ml for C. albicans, C. glabrata, C. tropicalis, and
C. parapsilosis, respectively. Marcos-Arias et al. (2012) previ-
ously showed that the GM MICs for POS were 0.036 𝜇g/ml for
C. parapsilosis, 0.062 𝜇g/ml for C. albicans, 0.085 𝜇g/ml for C.
tropicalis, and 0.498 𝜇g/ml for C. glabrata [16]. Several other
studies also demonstrated that POS and VRC were strong
antifungal agents against Candida spp. [40–44].
In our study, all non-albicans Candida isolates were
susceptible to POS, although only 88% these isolates were
susceptible to VRC. In line with the Marcos-Arias et al. (2012),
VRC was effective against 95.5% of strains [16]. In addition,
the GM MICs for ITC were 0.04 𝜇g/ml for C. albicans,
0.26 𝜇g/ml for C. glabrata, 0.27 𝜇g/ml for C. tropicalis, and
0.46 𝜇g/ml for C. parapsilosis. Other studies have shown that
ITC is useful for treating patients with DS [27, 45, 46].
Dorocka-Bobkowska and Konopka (2007) reported that
AMB, FLC, and ITC were effective against 100%, 88.7%,
and 87.3% of C. albicans and 79.6%, 71.4%, and 79.6% of
other Candida strains, respectively [10]. In the present study,
AMB, FLC, and ITC were effective against 100%, 84.5%,
and 100% of C. albicans and 86%, 88%, and 80% of non-
albicans Candida isolates, respectively. Caspofungin is known
as an echinocandin fungicidal antifungal agent against most
Candida spp. [15].
Some data are available on the AFST of Candida spp. iso-
lated from denture-associated stomatitis (DAS) to echinocan-
dins [15, 47]. In the present study, only 2 isolates (2.3%) of the
84 isolates of C. albicans were resistant to CAS. We also found
that 14 isolates (28%) of the non-albicans Candida strains
were resistant to CAS.
In the present study, TRB was not found to be effective
against Candida spp. Ryder et al. (1998) also reported that
TRB was not an active drug against C. glabrata and C.
tropicalis [25].
Our results revealed that the tested antifungal showed
good activity for most isolates; however, variability observed
among some isolates and resistance to drugs highlight the
need for AFST as a monitor to management of therapeutic
procedure.
5. Conclusion
In conclusion, all Candida spp. isolated from patients wearing
dentures were susceptible to POS and AMB. As an antifungal,
POS could be a suitable alternative to the present antifungal
agents used for the management of CADS and could be also
used in the treatment of recurrent candidiasis.
Data Availability
The data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
The authors of this paper reported no conflicts of interest.
BioMed Research International
Authors’ Contributions
Dr. Saeid Mahdavi Omran (supervisor) conceived and
designed the experiments. Dr. Mojtaba Taghizadeh Armaki
performed the experiments. Vahid Moqarabzadeh analyzed
the data. DD. Maryam Zuashkiani and Maryam Rezaie
Dastjerdi conducted the sampling procedure. All authors
helped to write the paper.
Acknowledgments
This work was supported by the Infection Diseases Research
Center (IDRC), Babol University of Medical Sciences
(BUMS), Babol, Iran (Contract no. IRCT2013042713136N1).
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